Serms Promote Anti-Inflammatory Signaling and Phenotype of CD14+ Cells
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Inflammation ( # 2018) DOI: 10.1007/s10753-018-0763-1 ORIGINAL ARTICLE SERMs Promote Anti-Inflammatory Signaling and Phenotype of CD14+ Cells Lauri Polari ,1,4 Anu Wiklund,1 Sofia Sousa,1,2 Lauri Kangas,3 Tero Linnanen,3 Pirkko Härkönen,1 and Jorma Määttä1 Abstract— Signaling via estrogen receptors (ER) is recognized as an essential part of the immune regulation, and ER-mediated signaling is involved in autoimmune reactions. Espe- cially ERα activation in immune cells has been suggested to skew cytokine production toward Th2/M2-type mediators, which can have protective effect on inflammatory diseases and reduce Th1 and Th17 responses. These effects are caused by increased alternative activation of macrophages and changes in the activation of different T cell populations. In humans, hormonal status has been shown to have a major impact on several inflammatory diseases. Selective estrogen receptor modulators (SERMs) are ER ligands that regulate ER actions in a tissue-specific manner mostly lacking the adverse effects of steroid hormones. The impact of SERMs on the immune system is less studied, but it is suggested that certain SERMs may also produce immunoprotective effects. Here, we show that two novel SERMs and raloxifene affect immune cells by promoting M2 macrophage phenotype, alleviating NFκB activity, inhibiting T cell proliferation, and stimulating the production of anti- inflammatory compounds such as IL10 and IL1 receptor antagonist. Thus, these compounds have high potency as drug candidates against autoimmune diseases. KEY WORDS: estrogen receptor; SERM; macrophages; inflammation; raloxifene; estradiol. INTRODUCTION source of immune stimulus, and variability of expression of ER subtypes in the cellular microenvironment [7]. Estro- Signaling via estrogen receptors (ER) is recognized as genic compounds such as female sex hormones elicit their an essential part of the immune regulation, and ER- effects via ER. Upon ligand binding, ER initiates gene mediated signaling involved in both chronic inflammatory transcription in the nuclei and also elicits immediate effects diseases and autoimmune reactions [1–6]. This regulation via cytosolic signaling cascades. ER have been utilized as a can be either pro- or anti-inflammatory depending on sev- drug target for several estrogen-regulated diseases, most eral criteria such as types of organs and cells involved, importantly breast cancer and osteoporosis, in estrogen- sensitive organs [8]. However, ER-modulated inflammato- ry diseases and autoimmune reactions are not only limited Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10753-018-0763-1) contains supplementary ma- to traditional estrogen target tissues. terial, which is available to authorized users. Estrogenic signaling is suggested to affect immunomodulation in a wide array of inflammatory dis- 1 Institute of Biomedicine, University of Turku, Turku, Finland 2 Faculté de Médecine, Université Lyon-1, Lyon, France eases such as intestinal inflammation and CNS autoimmu- 3 Forendo Pharma Ltd., Turku, Finland nity [7]. ER ligands possibly induce anti-inflammatory 4 To whom correspondence should be addressed at Institute of Biomedi- effects via mechanisms involving ERα and GPER cine, University of Turku, Turku, Finland. E-mail: [email protected] 0360-3997/18/0000-0001/0 # 2018 The Author(s) Polari, Wiklund, Sousa, Kangas, Linnanen, Härkönen, and Määttä activation on immune cells, inducing a Th2-type skew in patterns of cytokine production and receptor expression, the cytokine milieu and reducing Th1 and Th17 responses according to their microenvironmental cues [24]. Several [1, 9–13]. This anti-inflammatory shift includes increased studies suggest that E2 activation modulates macrophage M2 characteristics in monocyte macrophage populations phenotype and function [12, 16, 25–28] although E2 does and changes in the activity and number of T regulatory not elicit strong proliferative response in myeloid cells in a cells (Treg) [14–17]. It is intriguing that a similar Th2-type similar degree as in classical estrogen-sensitive tissues skew in inflammatory mediators has been observed to such as breast epithelium and endometrium [19]. Never- occur at the third trimester of pregnancy—aperiodalso theless, recent studies suggest that endogenous E2 may characterized by increased estrogen levels [18]. These ob- also induce macrophage activity and self-renewal in vivo servations suggest that ER signaling regulates the immune [16, 26, 29]. The expression of ER in myeloid cells has system cells by modulating their responses to inflammato- been demonstrated, but the pattern of ER subtypes and ry stimuli. splice variants in macrophages and their possible contribu- The activation of ER signaling is considered to stim- tion to the innate immunity is likely to be context/tissue ulate anti-inflammatory response. Accordingly, 17β- specific [16, 28, 30, 31]. estradiol (E2), a strong ER agonist steroid hormone, is We have studied the immune-modulatory properties associated with amelioration of inflammatory diseases of SERM drugs and several novel SERM candidates and [7]. E2 is not, however, utilized as an immunomodulatory examined their effects on NFκB activity and macrophage drug because it may increase a risk for tumor formation in surface protein expression in human monocytes. Among estrogen-sensitive tissues [19]. Therefore, it is not an opti- those compounds, we have selected the most M2-type mal drug for long-term medication. Instead, a recent clin- activation promoting compounds to be further studied in ical trial suggests that another natural estrogen hormone, models of macrophage differentiation and cytokine pro- estriol, may be more suitable as an immune modulator. duction. Here, we present evidence that specific SERM Estriol, the natural level of which is high especially during compounds can modulate the function of the monocyte pregnancy, together with glatiramer acetate, has been macrophage cell population. We show that specific SERMs shown to reduce the relapse rate of female MS patients can modulate the differentiation of CD14-positive cells by [14, 20]. This result is supported by clinical findings show- skewing their phenotype toward M2 macrophages and ing that symptoms of multiple sclerosis are often alleviated inducing the production of anti-inflammatory cytokines. during pregnancy [21]. These novel, previously unpublished SERM compounds Selective estrogen receptor modulators (SERMs) are promoted M2-like macrophage activation in pharmacolog- compounds that elicit estrogenic and/or antiestrogenic ef- ical concentration. They could thus be beneficial for the fects in a tissue-specific manner. The first non-steroidal alleviation of systemic inflammation in autoimmune SERM developed for clinical use was tamoxifen, which diseases. has been used as an endocrine therapy for breast cancer since the late 1980s [22]. Thereafter, a few other SERM drugs such as raloxifene, toremifene, and ospemifene have MATERIALS AND METHODS been developed for treatment and/or prevention of various diseases in estrogen-responsive tissues. As the activation of SERMs ERsinimmunecellshasbeenlinkedwithdownregulation All SERM compounds, including novel compounds of inflammation, it is tempting to expect that an appropriate investigated in this study, were a kind gift from Forendo SERM might regulate the ER signaling in immune cells but Pharma Ltd., Turku, Finland, unless stated otherwise. Ral- not induce adverse effects in other estrogen-sensitive oxifene and 17β-estradiol were purchased from Sigma (St. tissues. Louis, USA). Working dilutions of estrogenic compounds Monocytes and macrophages are myeloid antigen- were prepared in dimethylsulfoxide and kept at − 20 °C. presenting cells that have a central role in the initiation of immune responses thus possessing a major role in adaptive Estrogen Receptor Affinity Assay immunity. In addition to phagocytosis, they can either promote or alleviate immune responses [23]. Currently, it The binding affinity of SERM2, SERM7, and ralox- is widely accepted that rather than just pro-inflammatory ifene to ERα and their ability to compete in binding with M1 and anti-inflammatory M2 dichotomy, the macro- E2 (incubation time 2 h, estrogen concentration 2 nM) phages can adapt a spectrum of phenotypes with different were determined using a commercially available assay kit SERMs Promote Anti-Inflammatory Signaling and Phenotype of CD14+ Cells according to the manufacturer’s instructions (PanVera (IL4) (50 ng/ml), or IL10 (50 ng/ml) [36]. M(IFNγ)cells LCC, Madison, WI) [32]. The IC50 values were obtained were activated with lipopolysaccharide (LPS, 10 ng/ml) by fitting the data to the Hill’sequation[33]. after 5-day incubation with IFNγ. Monocytes were treated with SERMs or E2 to assess Cell Culture their effects on macrophage polarization and phenotype. Compounds were added 4 days after the differentiation Human THP-1 Lucia NFκB reporter cells were pur- stimuli (IFNγ), followed by LPS addition 24 h later. After chased from InvivoGen (San Diego, CA, USA). The THP- 6 days of culturing, medium samples were collected and 1LuciaNFκB cell line contains a stable NFκB inducible cells were either lyzed for RNA extraction with RA1 lysis Lucia reporter construct which is secreted to the growth buffer (Macherey-Nagel, Duren, Germany) supplemented medium. THP-1 Lucia cells were grown in a colorless with β-mercaptoethanol or detached (with Accutase) for RPMI-1640 growth medium with 4.5 g/l glucose,